This grant proposes to use patch-clamp recording, Ca2+ fluorometric methods, and tissue culture to study the physiology and pharmacology of epithelial cells from the ciliary body. We are proposing to make whole- cell and perforated patch recordings from pigmented and nonpigmented cells under voltage clamp and to study changes in conductance produced by substances known to have some pharmacological effect on these cells (bombesin, carbachol, vasopressin, isoproterenol, phenylephrine, ATP, VIP) or to have effects on the intraocular pressure (catecholamines, dopamine, carbonic anhydrase inhibitors, phorbol esters, forskolin). When changes in membrane conductance can be recorded, the effects of the drugs will be characterized as to their ion selectivity, voltage dependence, and mechanism of action (role of G proteins, second messengers). We are also proposing to use the fluorometric dye fura-2 to study a possible role of Ca2+ in the response of the nonpigmented ciliary body epithelial cells to pharmacological agents, both in intact ciliary processes and for cells in primary culture. Simultaneous measurements will be made of changes in Ca2+ concentration and changes in membrane conductance, to test critically the notion that Ca2+ acts as a second messenger in this tissue. We are proposing to develop a method for culturing monolayers of nonpigmented cells on permeable supports. Our goal is to produce monolayers with sufficiently high transepithelial resistances that they can be used to make measurements of ion and water flux in this tissue. If we are able to produce such monolayers, we shall use them to study transepithelial electrical parameters (open circuit voltage and closed circuit current), and the flux of ions (Na+,K+,Cl-) which are likely to be important in the secretion of the aqueous humor. We hope also to make quantitative measurements of water flux across monolayers of nonpigmented cells, to investigate the mechanisms and pharmacology of water transport in the ciliary body.